Neospora caninum is an intracellular parasitic protozoon using an animal belonging to the canine family as a final host and cattle, sheep, a goat, deer, or the like as an intermediate host. The Neospora is transmitted by horizontal infection from oocyst discharged in the feces of a final host or by vertical infection in an intermediate host and, particularly in cattle, causes miscarriage, stillbirth, or abnormal labor mainly characterized by neurological symptoms in calf at a high rate. Neospora caninum infection is estimated to account for about 40% of bovine miscarriage cases in reports from Western countries. The vertical infection with Neospora caninum has been established over repeated generations, which is cited as a biggest factor in the spread of infection with the protozoon. In fact, cases of infection with Neospora caninum have been reported all over the world, and its occurrence is also serious in Japan. Bovine dams infected with Neospora caninum cannot often be milked, leading to the wastage of calves and the milking amount and the like; thus, its occurrence causes remarkably large economic loss in the livestock industry. There is also an estimation that global damage due to Neospora caninum infection annually aggregates about billions of US dollars.
The following needs to be considered for a vaccine for preventing the Neospora caninum infection. Because Neospora caninum is an intracellular parasitic protozoon, cellular immunity is important for protective immunity thereagainst. Particularly, the function of interferon gamma and CD4-positive T cells is important. The effect of antibody on Neospora caninum is a controversial subject; however, the antibody is believed to react to Neospora caninum present outside the cells to prevent the spread thereof in the body.
Infection during pregnancy causes miscarriage or vertical infection due to the characteristic immune response of the mother's body. That is, when Neospora caninum infection occurs during early pregnancy, miscarriage is induced by the immune reaction of the mother's body. When Neospora caninum infection occurs during mid-pregnancy during which the mother's body is immunosuppressed, vertical infection to the fetus is established, and an individual congenitally infected with Neospora caninum is born.
Some vaccines against Neospora caninum using inactivated and live forms of the protozoon have previously been attempted to be developed on bovine neosporosis. The use of the inactivated Neospora caninum and POLYGEN as an adjuvant could not prevent the infection of fetuses therewith (Non Patent Literature 1). The use of the inactivated Neospora caninum and HAVLOGEN as an adjuvant is reported to have a prophylactic effect of 5.2% to 54% in New Zealand and of 46% in Costa Rica (Non Patent Literature 1). Vaccination with the live vaccine is also reported to have the effect of reducing stillbirth. However, the effect of these vaccines on naturally-infected cattle has not yet been demonstrated.
In addition to the inactivated and live vaccines, recombinant vaccines targeting proteins derived from Neospora caninum have been attempted to be prepared. Experiments using a mouse model resulted in the vertical infection thereof with the protozoon being suppressed by recombinant vaccines using Neospora caninum-derived proteins: MIC1, MIC3, GRA2, GRA6, SAG1 and SRS2 (Non Patent Literatures 2 and 3).
However, vectors derived from a microorganism and a virus were used for the preparation of the above recombinant vaccines; thus, the pathogenicity of these vectors is a problem in vaccinating an animal therewith. Accordingly, to prepare a recombinant vaccine, it represents a challenge to develop a vector and an adjuvant high in safety and excellent in the ability to induce cellular immunity.
Liposomes coated with a macromolecular polysaccharide such as mannan developed as an adjuvant for vaccines and immunotherapy are reported to have a strong ability to induce cellular immunity (Patent Literature 1 and Non Patent Literature 4). However, mannan is a mixture of polymannoses with heterogeneous molecular weights and also has strong toxicity to a living body (Non Patent Literature 5); thus, it is not suitable for a pharmaceutical product.
Meanwhile, Mizuochi et al. reports that an antigen is included in liposomes whose surface has an oligosaccharide comprising 2 to 11 sugar residues and binding to a lectin derived from antigen-presenting cells to remove the toxicity and antigenicity of the sugar and enhance its effect as a vaccine (Patent Literature 2). This document also discloses that cellular immunity against the antigen included in the liposomes each having the oligosaccharide on the surface can be efficiently induced. The liposome having the oligosaccharide on the surface is thought to be phagocytized by antigen-presenting cells via mannose receptors, resulting in the presentation of the antigen through an MHC class I or II molecule for the activation of antigen-specific T cells and the induction of Th1-derived cytokines.
Shimizu et al. report that when a Leishmania major-derived soluble antigen is included in liposomes each having an oligosaccharide on the surface and mice are immunized with these liposomes, Th1-type immunity against Leishmania major is significantly induced to control the infection of the mice with the protozoon (Non Patent Literature 6).